Invasive aspergillosis (IA), caused by the fungus Aspergillus fumigatus, is a leading cause of death in immunocompromised patients. Current IA therapy has a disappointing treatment success rate. Our long-term goal is to elucidate the cellular and molecular mechanisms required for A. fumigatus pathogenesis. IA occurs when A. fumigatus spores germinate into invading hyphae. Our objective is to identify and characterize key downstream targets of the calcineurin pathway that specifically regulate A. fumigatus hyphal growth. Our central hypothesis is that the calcineurin pathway controls critical steps in cell wall formation and hyphal growth required for disease. Once this molecular control of cell wall homeostasis and hyphal growth is understood, targeted inhibition of these processes can lead to the development of novel diagnostic markers and antifungal treatments. Based on our molecular model and collection of mutants, we hypothesize that there are effectors of the calcineurin pathway that specifically regulate A. fumigatus hyphal growth. We will identify these hyphal growth regulators using transcriptional profiling, regulatable expression, and targeted mutagenesis with animal models to determine their roles in hyphal growth and pathogenicity. We also hypothesize that specific calcineurin pathway proteins interact with and control cell wall and hyphal growth proteins. We will purify and tag calcineurin pathway proteins and characterize their binding to effectors by affinity chromatography and differential dephosphorylation to elucidate interactions controlling the mechanism of calcineurin's regulation over hyphal growth. We further hypothesize that there are critical domains of key calcineurin pathway protein that regulate cell wall formation and hyphal growth. We will generate targeted protein mutations and analyze biochemical cell wall and hyphal phenotypes, localization, and phosphatase activity to characterize the specific function of key calcineurin pathway component domains. The expected outcome is to understand the molecular mechanism surrounding hyphal growth to provide critical knowledge needed for strategies to target specific calcineurin pathway components for both novel diagnostic markers and antifungal treatments. PUBLIC HEALTH RELEVANCE: Invasive fungal infections are a leading cause of death for patients with lowered immune systems. A public health task force identified Aspergillus fumigatus as one of six infections where a breakthrough was urgently needed. Despite a high mortality, the fundamental mechanism A. fumigatus uses to cause disease is not understood and this knowledge is critical to improving diagnosis and treatment for this deadly disease.